VEGFR2 induces c-Src signaling and vascular permeability in vivo via the adaptor protein TSAd

Regulation of vascular endothelial (VE) growth factor (VEGF)-induced permeability is critical in physiological and pathological processes. We show that tyrosine phosphorylation of VEGF receptor 2 (VEGFR2) at Y951 facilitates binding of VEGFR2 to the Rous sarcoma (Src) homology 2-domain of T cell-specific adaptor (TSAd), which in turn regulates VEGF-induced activation of the c-Src tyrosine kinase and vascular permeability. c-Src was activated in vivo and in vitro in a VEGF/TSAd-dependent manner, and was regulated via increased phosphorylation at pY418 and reduced phosphorylation at pY527. Tsad silencing blocked VEGF-induced c-Src activation, but did not affect pathways involving phospholipase Cγ, extracellular regulated kinase, and endothelial nitric oxide. VEGF-induced rearrangement of VE-cadherin-positive junctions in endothelial cells isolated from mouse lungs, or in mouse cremaster vessels, was dependent on TSAd expression, and TSAd formed a complex with VE-cadherin, VEGFR2, and c-Src at endothelial junctions. Vessels in tsad(-/-) mice showed undisturbed flow and pressure, but impaired VEGF-induced permeability, as measured by extravasation of Evans blue, dextran, and microspheres in the skin and the trachea. Histamine-induced extravasation was not affected by TSAd deficiency. We conclude that TSAd is required for VEGF-induced, c-Src-mediated regulation of endothelial cell junctions and for vascular permeability.     

Priming effect of homocysteine on inducible vascular cell adhesion molecule-1 expression in endothelial cells

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis, as well as for arterial and venous thrombosis. However, the mechanisms through which elevated circulating levels of homocysteine cause vascular injury and promote thrombosis remain unclear. Here, we tested the hypothesis that homocysteine (Hcy) sensitizes endothelial cells to the effect of inflammatory mediators. Human umbilical vein endothelial cells (HUVEC) were incubated with Hcy 1.0 mM for varying time points, and then treated in the absence or presence of 1.5 U/ml thrombin or 10 mg/ml lipopolysaccharide (LPS). Hcy alone had no effect on the expression of vascular cell adhesion molecule (VCAM)-1. However, Hcy enhanced thrombin- and LPS-mediated induction of VCAM-1 mRNA and protein levels. Consistent with these results, pretreatment of HUVEC with Hcy resulted in a two-fold increase in LSP-mediated induction of leukocyte adhesion. The latter effect was significantly inhibited by anti-VCAM-1 antibodies. Together, these findings suggest that Hcy sensitizes HUVEC to the effect of inflammatory mediators thrombin and LPS, at least in part through VCAM-1 expression and function.     

Eosinophilic perifolliculitis presenting as a painful cystic ovarian mass in a woman with fibromyalgia: a case report

BACKGROUND: Autoimmune oophoritis is characterized by an ovarian lymphocytic infiltrate and is a rare finding in women with premature ovarian failure. Eosinophilic perifolliculitis is a possible variant of autoimmune oophoritis, of which the pathogenesis and natural history are largely unknown. CASE: A 45-year-old woman, gravida 2, para 2, status post total abdominal hysterectomy, presented to her internist complaining of cyclic, throbbing, right lower quadrant pain. Her past medical history was significant forfibromyalgia. Pelvic ultrasound demonstrated a 2.3-cm, physiologic-appearing right ovarian cyst. Follow-up ultrasound showed a 2.2-cm, complex cyst on the right ovary that increased in size to 4.2 x 3.2 x 3.5 cm on repeat ultrasound 12 weeks later. Exploratory laparotomy and bilateral salpingo-oophorectomy were performed. Pathologic evaluation of the ovaries revealed a 3 x 2 cm regressing corpus luteal cyst with numerous eosinophils, lymphocytes, macrophages and plasma cells, infiltrating the cyst zoall. Serum antiovarian antibodies were positive. CONCLUSION: The patient's pathologic findings are consistent with the rare entity of eosinophilic perifolliculitis. The patient's history offibromyalgia is of particular interest given that both of these diseases may have an autoimmune etiology. Eosinophilic perifolliculitis should be considered in the differential diagnosis of premenopausal and perimenopausal women with pelvic pain and persistent cystic ovarian enlargement.     

Use of X-chromosome inactivation pattern to determine the clonal origins of uterine leiomyoma and leiomyosarcoma

Uterine leiomyomas (LMs) and leiomyosarcomas (LMSs), both of smooth muscle origin, sometimes coexist in the same uterus. Little genetic evidence exists concerning the developmental relationship between LM and LMS. Using the X-chromosome inactivation pattern of the human androgen receptor gene, we examined the clonality of LM and LMS. Of the 24 patients with LM, 21 had multiple neoplasms; all were clonal and individual LMs derived from separate clones. Of the 20 patients with LMS, 6 exhibited multiple tumors in the uterus, and 4 of these individuals also harbored coexisting uterine LMs. We found all LMSs to be clonal. Separate tumors showed identical pattern of X inactivation in 4 patients, and in 2 other individuals, multiple LMSs developed from independent clones. Among the 4 patients with LMS and coexisting LM, 3 showed the same pattern of X inactivation in LMS and the adjacent LM. In 2 of the 3 patients, the tumor also exhibited a morphological transition between benign cells in LM and malignant cells in LMS, supporting the possibility of transformation from LM to LMS. One patient displayed different clones in LMS and the coexisting LM, indicating their independent origins. We conclude that (i) both LM and LMS are clonal; (ii) different nodules in multiple LM are of independent origins; (iii) multiple lesions of LMS may be either monoclonal or multiclonal; (iv) most LMSs are solitary lesions and are most likely de novo, but an individual LM may undergo "malignant transformation" to a LMS; and (v) some LMSs and coexisting LMs are of independent origins.     

High nuclear grade, frequent mitotic activity, cyclin D1 and p53 overexpression are associated with stromal invasion in mammary intracystic papillary carcinoma

Stromal invasion is identified with difficulty in routine hematoxylin-eosin-stained sections of core needle biopsy specimens from mammary intracystic papillary carcinomas. The goal of this study was to determine if nuclear grade, mitotic activity, and immunohistochemical stains for p53 and cyclin D1 would assist in differentiating intracystic papillary carcinomas without stromal invasion (ICPC) from tumors with stromal invasion (ICPC-INVA). Eight cases of ICPC and 12 cases of ICPC-INVA were reviewed. Hematoxylin-eosin slides were examined to determine the histologic features. Immunohistochemistry was performed using monoclonal antibodies to human p53 and cyclin D1. Fisher's exact test was used to compare the nuclear grade, mitotic activity, and immunoreactivity between ICPC and ICPC-INVA. High nuclear grade was more often associated with ICPC-INVA than with ICPC, although the difference was not statistically significant (p = 0.069). Frequent mitotic activity was associated with ICPC-INVA more than with ICPC (p = 0.0198). All cases of ICPC were negative for either p53 or cyclin D1, whereas 7 of 12 cases (58.3%) of ICPC-INVA were positive for either cyclin D1 alone (3 cases), p53 alone (3 cases), or both cyclin D1 and p53 (1 case) (p = 0.0147). Identical nuclear grade, mitotic activity, and immunostaining patterns were seen in the intracystic and the invasive components, and in the core biopsy and the excision of the same tumor. When any one of the positive indicators (high nuclear grade, frequent mitotic activity, or positive immunostains for cyclin D1 and/or p53) was present, the positive predictive value for stromal invasion was 91.7%. When none of the positive indicators was present, the negative predictive value was 87.5%.     

The PLC-PKC cascade is required for IL-1beta-dependent Erk and Akt activation: their role in proliferation

We investigated the signaling mechanisms that lead to IL-1beta-induced cell proliferation. Treatment of Balb 3T3 cells with IL-1beta activated two signaling pathways, Erk and Akt. IL-1beta also increased tyrosine phosphorylation of PLC-gamma in Src kinase-dependent manner. Pharmacological inhibition of the PLC-PKC cascade by using specific inhibitor for PLC-gamma (U73122) and PKC (GFX) strongly inhibited IL-1beta-induced Erk and Akt activation. Inhibition of MEK1 by its specific inhibitor, PD98059 substantially inhibited Erk activation. Similarly, inhibition of PI3K activation by its specific inhibitor LY294002 suppressed Akt phosphorylation. Moreover, IL-1beta-induced association of PLC-gamma with SHPS-1. SHPS-1 mutants lacking the tyrosine phosphorylation sites failed to associate with PLC-gamma. Finally, IL-1beta-induced proliferation of Balb 3T3 cells and inhibition of Erk and Akt signalings or their upstream signaling molecules, Src kinase and PKC by their inhibitors strongly inhibited IL-1beta-dependent cell proliferation. Taken together, our results suggest that a SHPS-1-PLC-gamma complex activate the PLC-PKC cascade, which is required for the activation of IL-1beta-dependent Erk and Akt signalings and cell proliferation.     

SH2 domain containing protein tyrosine phosphatase 2 regulates concanavalin A-dependent secretion and activation of matrix metalloproteinase 2 via the extracellular signal-regulated kinase and p38 pathways

We investigated the role of SH2 domain containing protein tyrosine phosphatase (SHP) 2 in Concanavalin A (Con A) -dependent signaling that leads to the augmented secretion and activation of matrix metalloproteinase (MMP) 2. In cells expressing mutant SHP-2 in which 65 amino acids in the SH2-N domain were deleted, we found that production, secretion, and proteolytic activation of MMP-2 in response to Con A treatment was severely impaired. Under Con A stimulation, complex formation of SHP-2 with SOS-1 and Grb-2 together with the activation of Ras signaling was clearly observed in wild-type cells, but not in SHP-2 mutant cells. In wild-type cells, Con A-treatment activated dual signaling pathways, extracellular signal-regulated kinase (Erk) and p38, in a Ras-dependent manner, whereas Con A-dependent activation of these signaling pathways was absent in SHP-2 mutant cells. In addition, pretreatment of wild-type cells with U0126, a potent inhibitor for mitogen-activated protein/ERK kinase 1, or with SB203580, a specific inhibitor for p38, significantly inhibited the Con A-dependent secretion and activation of MMP-2. However, overexpression of active mitogen-activated protein/ERK kinase 1 in SHP-2 mutant cells could not induce clear activation of MMP-2 secretion, although these cells responded well to the Con A treatment in a p38-dependent manner. Finally, reintroduction of wild-type SHP-2 into SHP-2 mutant cells rescued Erk and p38 activation, and also MMP-2 secretion, whereas dominant-negative SHP-2 could block the Con A-dependent activation of Erk and p38. Taken together, our results strongly suggest that SHP-2 plays a critical role as a positive mediator for Con A-dependent activation of MMP-2 secretion via Ras-Erk and Ras-p38 signalings.